U.S. patent application number 13/497421 was filed with the patent office on 2012-08-02 for composition comprising s-allyl-l-cysteine as active ingredient for preventing or treating gastrointestinal disorders.
This patent application is currently assigned to PHARMAKING CO., LTD.. Invention is credited to Sung Hye Bang, Gwang Soon Kim, Soon Bae Kim, Wan Bae Kim, Wie Jong Kwak.
Application Number | 20120196936 13/497421 |
Document ID | / |
Family ID | 43796388 |
Filed Date | 2012-08-02 |
United States Patent
Application |
20120196936 |
Kind Code |
A1 |
Kim; Soon Bae ; et
al. |
August 2, 2012 |
Composition Comprising S-Allyl-L-Cysteine as Active Ingredient for
Preventing or Treating Gastrointestinal Disorders
Abstract
A composition including S-allyl-L-cysteine as an active
ingredient and having an anti-Helicobacter pylori activity or a
gastric mucosa protective effect, a composition for preventing,
relieving, or treating gastrointestinal disorders, and a method of
using the compositions.
Inventors: |
Kim; Soon Bae; (Gyeonggi-Do,
KR) ; Kim; Gwang Soon; (Gyeonggi-Do, KR) ;
Kim; Wan Bae; (Seoul, KR) ; Kwak; Wie Jong;
(Seoul, KR) ; Bang; Sung Hye; (Seoul, KR) |
Assignee: |
PHARMAKING CO., LTD.
Chungcheongbuk-Do
KR
|
Family ID: |
43796388 |
Appl. No.: |
13/497421 |
Filed: |
September 20, 2010 |
PCT Filed: |
September 20, 2010 |
PCT NO: |
PCT/KR2010/006506 |
371 Date: |
April 16, 2012 |
Current U.S.
Class: |
514/562 ;
562/557 |
Current CPC
Class: |
A61K 9/0019 20130101;
A61K 47/26 20130101; A61K 9/282 20130101; A61K 9/2853 20130101;
A61P 1/00 20180101; A61P 1/16 20180101; A23L 33/175 20160801; A61K
9/145 20130101; A61P 31/04 20180101; A61K 36/8962 20130101; A61K
9/146 20130101; A61P 25/06 20180101; A61P 37/08 20180101; A61P
15/08 20180101; A61K 9/08 20130101; A23V 2002/00 20130101; A61P
35/00 20180101; A61K 31/195 20130101; A61K 47/38 20130101; A61K
9/10 20130101; A61P 1/04 20180101; A61K 9/2054 20130101; A61K
31/198 20130101; A61K 9/2833 20130101; A61P 17/04 20180101; A61K
9/2018 20130101; A61K 9/0095 20130101; A61P 9/10 20180101; A23V
2002/00 20130101; A23V 2200/32 20130101; A23V 2250/0616
20130101 |
Class at
Publication: |
514/562 ;
562/557 |
International
Class: |
A61K 31/195 20060101
A61K031/195; A61P 35/00 20060101 A61P035/00; A61P 1/04 20060101
A61P001/04; C07C 323/58 20060101 C07C323/58; A61P 1/00 20060101
A61P001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 23, 2009 |
KR |
10-2009-0090232 |
Claims
1. A pharmaceutical composition comprising S-allyl-L-cysteine, a
pharmaceutically acceptable salt of S-allyl-L-cysteine, or a
solvate or hydrate thereof as an active ingredient and having an
anti-Helicobacter pylori activity.
2. The pharmaceutical composition of claim 1, wherein the
anti-Helicobacter pylori activity comprises activities that prevent
or treat hepatic encephalopathy, arteriosclerosis, hepatobiliary
system-associated diseases, urticaria, migraine, short stature,
infertility, food allergy, chronic gastritis, acute gastritis,
gastric ulcer, gastric cancer, bleeding in gastrointestinal tract,
gastroesophageal reflux disease, duodentis, or duodenum ulcer.
3. A pharmaceutical composition comprising S-allyl-L-cysteine, a
pharmaceutically acceptable salt of S-allyl-L-cysteine, or a
solvate or hydrate thereof as an active ingredient for preventing
or treating gastrointestinal disorders.
4. The pharmaceutical composition of claim 3, wherein the
gastrointestinal disorders are selected from the group consisting
of chronic gastritis, acute gastritis, gastric ulcer, gastric
cancer, bleeding in gastrointestinal tract, gastroesophageal reflux
disease, duodentis, and duodenum ulcer.
5. A pharmaceutical composition comprising S-allyl-L-cysteine, a
pharmaceutically acceptable salt of S-allyl-L-cysteine, or a
solvate or hydrate thereof as an active ingredient and having a
gastric mucosa protective effect.
6. The pharmaceutical composition of claim 1, wherein the
S-allyl-L-cysteine is isolated from a plant belonging to the Allium
genus and purified, synthesized, or prepared by fermentation.
7. A pharmaceutical formulation comprising a composition according
to claim 1, the pharmaceutical formulation selected from the group
consisting of a formulation for oral administration, a formulation
for mucosal administration, an injection formulation, a formulation
for inhalation, and a formulation for external application.
8. The pharmaceutical formulation of claim 7, wherein the
formulation for oral administration is selected from the group
consisting of hard and soft capsules, tablets, suspensions, powder,
suspended-release formulations, enteric formulations, granules,
oleosacchara, fine granules, pills, extracts, liquids, aromatic
waters, emulsions, syrups, elixirs, fluid extracts,
infusodecoctions, tinctures, medicated spirits, and infused
oils.
9. A food composition comprising S-allyl-L-cysteine, a salt of
S-allyl-L-cysteine, or a solvate or hydrate thereof as an active
ingredient and having an anti-Helicobacter pylori activity or a
gastric mucosa protective effect.
10. A food composition comprising S-allyl-L-cysteine, a salt of
S-allyl-L-cysteine, or a solvate or hydrate thereof as an active
ingredient for preventing, relieving, or treating gastrointestinal
disorders.
11. The food composition of claim 10, wherein the gastrointestinal
disorders are selected from the group consisting of chronic
gastritis, acute gastritis, gastric ulcer, gastric cancer, bleeding
in gastrointestinal tract, gastroesophageal reflux disease,
duodentis, and duodenum ulcer.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATION
[0001] This application claims the benefit of Korean Patent
Application No. 10-2009-0090232, filed on Sep. 23, 2009, in the
Korean Intellectual Property Office, the disclosure of which is
incorporated herein in its entirety by reference.
TECHNICAL FIELD
[0002] 1. Field of the Invention
[0003] The present invention relates to a composition having an
anti-Helicobacter pylori activity and a gastric mucosa protective
effect, a composition for preventing or treating gastrointestinal
disorders, and a method of using the compositions.
[0004] 2. Background Art
[0005] Gastrointestinal disorders are caused by a variety of
factors and are known to be caused by an imbalance between
aggressive factors, such as Helicobacter pylori, gastric acid,
pepsin, overwork, stress, and alcohol,and defensive factors, such
as mucus secretion, tissue-regenerative capability, and
anticoagulant activity. Gastritis caused by overwork, stress,
Helicobacter pylori infection, or the like is a common symptom but
can develop, if not treated, into chronic gastritis, gastric ulcer,
and, rarely, gastric cancer. A gastric mucosal lesion caused by
alcohol can be healed within several days by the removal of the
stimulus factor but may develop into gastrointestinal bleeding, a
gastric perforation, or the like (Taeyoung Oh, et al., J. Applied
Pharmacology, Vol. 5, pp 202-210, 1997). Various drugs such as
cimetidine, ranitidin, famotidine, omeprazole, or bismuth have been
used for treating gastrointestinal disorders. However, the relapse
rate after discontinuation of these drugs is very high, and thus
there is a need to develop a novel drug.
[0006] Helicobacter pylori is a gram-negative bacterium that
colonizes human gastric mucosa or mucus. It is recognized that
Helicobacter pylori infection is a significant contributory factor
in the development of most gastrointestinal tract-associated
disorders such as acutechronic gastritis, atrophic gastritis,
gastric ulcer, gastric cancer, and duodenal ulcers (Crowe, Curr
Opin Gastrenterol., 21(1), pp 32-38, 2005). Furthermore, it has
been reported that Helicobacter pylori infection is a risk factor
for hepatic encephalopathy, arteriosclerosis, and hepatobiliary
system-associated diseases in addition to digestive
system-associated diseases (Karahalil, et al., Curr Drug Saf, 2, pp
43-46, 2007; Scragg, et al., J. Epidemiol Community Health, 50(5),
pp 578-579, 1996). According to the US Centers for Disease Control
and Prevention (CDC), Helicobacter pylori infection may cause
chronic fatigue, urticaria, migraine, short stature, infertility,
food allergy, etc., which are all symptoms of `strange Helicobacter
syndrome`. In general, antibiotics are used to kill Helicobacter
pylori. However, Helicobacter pylori reinfection is common after
eradication thereof and a high-dose treatment is required for a
long period of time to completely remove the Helicobacter pylori.
Accordingly,the long-term use of high dose antibiotics may lead to
side effects and increase in antibiotic-resistant bacteria.
[0007] Recently, diverse research is being conducted into materials
that inhibit the growth of bacteria in food as a safe method to
treat Helicobacter pylori infection-associated diseases.
Lactobacillus-fermented milk using probiotics, egg-yolk-derived
immunoglobulin (IgY) including a Helicobacter pylori-neutralizing
antibody, and catechin contained in wine and green tea are
considered as effective substances against infection by
Helicobacter pylori (McMahon, et al., Aliment Pharmacol Ther 23(8),
pp 1215-1223, 2006; Sachdeva, et al., Eur J Gastroenterol Hepatol,
21(1), pp 45-53, 2009; Shin, et al., J Med Microbiol., 53(Pt 1), pp
31-34, 2004).
[0008] Meanwhile, garlic (Allium Sativum.L) that belongs to the
Allium genus has antimicrobial, antifungal, anti-oxidant, and
anti-cancer properties (Ankri, et al., Microbes Infect. 1(2), pp
125-129, 1999), prevents thrombosis, inflammation, and oxidative
stress of cells (Sener, et al., Mol Nutr Food Res., 51(11), pp
1345-1352, 2007), and thus has drawn attention. Garlic contains
various components including steroidal saponin such as eruboside-B
having antifungal and anti-cancer properties (Matsuura H, et al.,
Chem Pharm Bull (Tokyo), 36: 3659-3663, 1988), glycoside fractions
having a cholesterol-lowering effect (Slowing, et al., J Nutr.,
131, pp 994S-9S, 2001), nonsulfur compounds such as
.beta.-chlorogenine having a platelet aggregation inhibiting effect
(Rahman K, et al., J. Nutr. 2006), and various organosulfur
compounds. Examples of the organosulfur compounds contained in
plants belonging to the Allium genus are fat-soluble organosulfur
compounds such as S-allyl-L-cysteine sulfoxides (alliin),
Dially-disulfide (DADS), and Diallyl sulfide (DAS) and
water-soluble organosulfur compounds such as S-allyl-L-cysteine
(SAC) and S-allylmercaptocysteine (SAMC).
[0009] It has been reported that SAC, which is an active ingredient
of mature garlic, has an anti-oxidant activity that inhibits
arteriosclerosis and anticancer activity in some cancer cell lines
(Proceedings of the American Association for Cancer Research, 30, p
181, 1989). However, it has not been reported that SAC has
therapeutic effects on gastrointestinal diseases and an
anti-Helicobacter pylori activity.
DISCLOSURE OF INVENTION
Technical Problem
[0010] In general, antibiotics are used to kill Helicobacter
pylori. However, Helicobacter pylori reinfection is common after
eradication thereof and high-dose treatment is required for a long
period of time to completely remove the Helicobacter pylori.
Accordingly, side effects may occur and antibiotic-resistant
bacteria may increase due to the use of antibiotics. Therefore,
there is a need for an alternative drug other than antibiotics. The
present invention provides a drug having an anti-Helicobacter
pylori activity and a gastric mucosa protective effect. The present
invention also provides a safe composition for preventing,
relieving, or treating gastrointestinal disorders.
Solution to Problem
[0011] According to an aspect of the present invention, there is
provided a composition comprising S-allyl-L-cysteine (SAC), which
is a water-soluble organosulfur compound contained in a plant
belonging to the Allium genus, as an active ingredient and having
an anti-Helicobacter pylori activity and a mucosa protective
effect. According to another aspect of the present invention, there
is provided a composition including SAC as an active ingredient for
preventing, relieving, or treating gastrointestinal disorders.
ADVANTAEOUS EFFECTS OF INVENTION
[0012] The composition including SAC as an active ingredient
according to the present invention inhibits Helicobacter pylori
infection and protects against gastric lesions caused by
Helicobacter pylori. Accordingly, the composition may be used as an
anti-Helicobacter pylori drug.
[0013] The composition including SAC as an active ingredient
according to the present invention prevents and treats gastric
mucosal lesions caused by hydrochloric acid-ethanol, aspirin, or
indomethacin, and thus may be efficiently used to prevent, relieve,
or treat gastrointestinal disorders. The composition including SAC
as an active ingredient according to the present invention may be
used as a pharmaceutical composition or food composition.
BRIEF DESCRIPTION OF DRAWINGS
[0014] The above and other features and advantages of the present
invention will become more apparent by describing in detail
exemplary embodiments thereof with reference to the attached
drawings in which:
[0015] FIG. 1 shows average weights of animals of control and
experimental groups for the entire test period, wherein all values
are shown as averages;
[0016] FIGS. 2A and 2B show an effect of S-allyl-L-cysteine (SAC)
on average serum IgG antibody production of animals of control and
experimental groups measured after Helicobacter pylori infection,
wherein all values are shown as averages and standard errors, **:
P<0.01, and *: P<0.05;
[0017] FIG. 3 shows an effect of SAC on average serum TNF-.alpha.
of animals of control and experimental groups measured for an
entire 10-week test period, wherein all values are shown as
averages and standard errors;
[0018] FIG. 4 shows tissue pathological changes of animals of
control and experimental groups;
[0019] FIG. 5 shows tissue pathological changes and the number of
eosinophils of animals of control and experimental groups, wherein
all values are shown as averages and standard errors, **:
P<0.01, and *: P<0.05;
[0020] FIG. 6 shows tissue pathological changes (mitotic figures)
of animals of control and experimental groups, wherein all values
are shown as averages and standard errors;
[0021] FIG. 7 shows serum glutamic oxaloacetic transaminase (GOT)
levels of animals of control and experimental groups, wherein all
values are shown as averages and standard errors;
[0022] FIG. 8 shows serum glutamate pyruvate transaminase (GPT)
levels of animals of control and experimental groups, wherein all
values are shown as averages and standard errors;
[0023] FIG. 9 shows serum copper and zinc containing-superoxide
dismutase (Cu/Zn-SOD) levels of animals of control and experimental
groups, wherein all values are shown as averages and standard
errors;
[0024] FIG. 10 shows an effect of SAC on lengths of gastric lesions
of rats induced by hydrochloric acid-ethanol, wherein ** indicates
a significant difference from a vehicle control group G1 by
p<0.01, G1: vehicle control group (distilled water), G2: 100
mg/kg of SAC, G3: 200 mg/kg of SAC, G4: 400 mg/kg of SAC, and G5:
positive control group (55.6 mg/kg of Stillen.RTM. as an active
ingredient);
[0025] FIG. 11 shows gastric lesion inhibiting rates in rats in
hydrochloric acid-ethanol-induced gastric lesion model, wherein **
indicates a significant difference from a vehicle control group G1
by p<0.01, G1: vehicle control group (distilled water), G2: 100
mg/kg of SAC, G3: 200 mg/kg of SAC, G4: 400 mg/kg of SAC, and G5:
positive control group (55.6 mg/kg of Stillen.RTM. as an active
ingredient);
[0026] FIG. 12 shows photographs of stomachs of rats in
hydrochloric acid-ethanol-induced gastric lesion model, wherein G1:
vehicle control group (distilled water), G2: 100 mg/kg of SAC, G3:
200 mg/kg of SAC, G4: 400 mg/kg of SAC, and G5: positive control
group (55.6 mg/kg of Stillen.RTM. as an active ingredient);
[0027] FIG. 13 shows an effect of SAC on an area of gastric lesions
of rats induced by aspirin, wherein *** indicates a significant
difference from a vehicle control group G1 by p<0.001, G1:
vehicle control group (distilled water), G2: 100 mg/kg of SAC, G3:
200 mg/kg of SAC, G4: 400 mg/kg of SAC, and G5: positive control
group (55.6 mg/kg of Stillen.RTM. as an active ingredient);
[0028] FIG. 14 shows gastric lesion inhibiting rates in rats in an
aspirin-induced gastric lesion model;
[0029] FIG. 15 shows photographs of stomachs of rats in an
aspirin-induced gastric lesion model;
[0030] FIG. 16 shows an effect of SAC on lengths of gastric lesions
of rats induced by indomethacin, wherein * indicates significant
difference from a vehicle control group G1 by p<0.05, G1:
vehicle control group (distilled water), G2: 100 mg/kg of SAC, G3:
200 mg/kg of SAC, G4: 400 mg/kg of SAC, and G5: positive control
group (55.6 mg/kg of Stillen.RTM. as an active ingredient);
[0031] FIG. 17 shows gastric lesion inhibiting rates in rats in an
indomethacin-induced gastric lesion model; and
[0032] FIG. 18 shows photographs of stomachs of rats in an
indomethacin-induced gastric lesion model.
DETAILED DESCRIPTION OF THE INVENTION
Mode for the Invention
[0033] Hereinafter, the present invention will now be described
more fully with reference to the accompanying drawings, in which
exemplary embodiments of the invention is shown.
[0034] A composition including S-allyl-L-cysteine (SAC) has an
excellent anti-Helicobacter pylori activity and gastric mucosa
protective effect.
[0035] The present inventors have found that a positive control
group of mice to which Helicobacter pylori is administered has a
significantly greater antibody titer (anti-H. pylori IgG) compared
to a negative control group of mice to which Helicobacter pylori is
not administered (p<0.01), but an experimental group to which
SAC and Helicobacter pylori are administered has a significantly
less Helicobacter pylori IgG compared to the positive control group
(FIGS. 2A and 2B). This result indicates that SAC has an effect of
inhibiting mice from being infected with Helicobacter pylori. The
present inventors have also found that the amount of TNF-.alpha.,
which is an inflammation factor associated with T cells, increased
in mice of the positive control group to which Helicobacter pylori
was administered but was reduced if SAC was administered with
Helicobacter pylori (FIG. 3). As a result, it can be seen that SAC
inhibits inflammation caused by Helicobacter pylori infection in
mice.
[0036] In order to observe a SAC effect on gastric lesions caused
by Helicobacter pylori infection, sections of mouse stomach tissue
were stained with hematoxylin and eosin (H&E staining). As a
result, in groups to which Helicobacter pylori was administered,
denaturation of gastric mucosa cells and eosinophil infiltration in
lamina propria were observed (FIG. 4), and the number of
eosinophils infiltrated into gastric mucosal epithelium in a
positive control group that was infected with Helicobacter pylori
was greater than that of a negative control group that was not
infected (p<0.01) but the number was significantly reduced in
the experimental group to which SAC was administered (p<0.05,
FIG. 5). The number of mitotic figures that represent cell nuclei
undergoing cell division was also increased in the Helicobacter
pylori-infected groups compared to the negative control group but
reduced in SAC-administered groups (FIG. 6). As a result, it can be
seen that gastric lesions caused by Helicobacter pylori can be
prevented or healed by SAC.
[0037] As a result of analyzing effects of SAC on serum biochemical
levels of animals, glutamic oxaloacetic transaminase (GOT) and
glutamate pyruvate transaminase (GPT) levels were the lowest in the
negative control group that was not infected, and the GOT and GPT
level of the SAC-administered group was less than the positive
control group (FIGS. 7 and 8).
[0038] Copper and zinc containing-superoxide dismutase (Cu/Zn-SOD)
level in serum was measured in order to observe effects of SAC on
oxidative lesions. As a result, the Cu/Zn-SOD levels were increased
in the Helicobacter pylori-infected groups compared to the negative
control group that was not infected. However, the Cu/Zn-SOD level
of the experimental group to which SAC were administered with was
higher than that of the positive control group to which
Helicobacter pylori was administered (FIG. 9). It was identified
that SOD that was expressed by a defense mechanism against
Helicobacter pylori infection was increased by SAC.
[0039] In addition, the present inventors have found that SAC has a
significant effect on inhibiting gastric mucosal lesions that were
induced in the rats by a drug administration. In a gastric lesion
induced in a rat by hydrochloric acid-ethanol, SAC exhibited a
significant reduction in the lesion when compared to a control
group to which only a vehicle was administered (FIGS. 10 and 12)
and a gastric lesion inhibiting rate (%) up to about 66% (FIG. 11).
SAC also exhibited a significant reduction in a gastric lesion
induced by aspirin (FIGS. 13-15) or indomethacin (FIGS. 16-18) when
compared to the negative control group. SAC has a gastric lesion
inhibiting rate (%) up to about 85% in the gastric lesion induced
by aspirin and a gastric lesion inhibiting rate (%) up to about 94%
in the gastric lesion induced by indomethacin, which are similar or
better gastric mucosa protective effects compared to Stillen.RTM.
used as a positive control group. The results indicate that SAC has
diverse gastric mucosa protective effects on gastric lesion caused
by various factors.
[0040] The present invention provides a pharmaceutical composition
including SAC as an active ingredient with a pharmaceutically
acceptable carrier, determined to have an anti-Helicobacter pylori
activity based on the results of experiments.
[0041] The present invention provides a pharmaceutical composition
including SAC as an active ingredient with a pharmaceutically
acceptable carrier and having a gastric mucosa protective
effect.
[0042] The present invention provides a food composition including
SAC as an active ingredient and having an anti-Helicobacter pylori
activity and a gastric mucosa protective effect.
[0043] The present invention provides a method of inhibiting
Helicobacter pylori infection and preventing gastric lesions caused
by Helicobacter pylori infection by using a composition including
SAC as an active ingredient.
[0044] The present invention provides a pharmaceutical composition
including SAC as an active ingredient and a pharmaceutically
acceptable carrier for preventing or treating gastrointestinal
disorders.
[0045] The present invention provides a food composition including
SAC as an active ingredient for preventing or relieving
gastrointestinal disorders.
[0046] The composition according to the present invention may
further include a therapeutic agent for gastrointestinal disorders
or an anti-Helicobacter pylori agent in addition to SAC.
[0047] The present invention provides a method of preventing or
treating gastrointestinal disorders using a composition including
SAC as an active ingredient and a pharmaceutically acceptable
carrier.
[0048] Helicobacter pylori infection or gastrointestinal disorders
are also common in animals, and thus the present invention also
provides a composition for animals.
[0049] The gastrointestinal disorders to which the composition
according to the present embodiment may be applied include chronic
gastritis, acute gastritis, gastric ulcer, gastric cancer, bleeding
in the gastrointestinal tract, gastroesophageal reflux disease
(GERD), duodentis, and duodenum ulcers, but are not limited
thereto.
[0050] The anti-Helicobacter pylori activity may include preventing
or treating hepatic encephalopathy, arteriosclerosis, hepatobiliary
system-associated diseases, urticaria, migraine, short stature,
infertility, food allergy, chronic gastritis, acute gastritis,
gastric ulcer, gastric cancer, bleeding in gastrointestinal tract,
gastroesophageal reflux disease (GERD), duodentis, or duodenum
ulcer, but is not limited thereto.
[0051] In the composition according to the present embodiment, a
pharmaceutically or sitologically acceptable salt of SAC may be
used as an active ingredient. The salt may be an acid addition salt
or a quaternary ammonium salt. Examples of the acid addition salt
include inorganic acid addition salts such as chloride,
hydrobromide, hydroiodide, sulfate, and phosphate and organic acid
addition salts such as oxalate, maleate, fumarate, lactate, malate,
succinate, tartrate, benzoate, and methanesolfonate. Examples of
the quaternary ammonium salt are a short-chain alkyl halogenide
such as methyl iodide, methyl bromide, ethyl iodide, and ethyl
bromide; a short-chain alkyl sulfonate such as methyl
methanesulfonate and ethyl methanesulfonate; and a short-chain
alkyl arylsulfonate such as methyl-p-toluenesulfonate.
[0052] The SAC or the pharmaceutically or sitologically acceptable
salt thereof may exist in a solvate or hydrate form, and thus a
solvate or hydrate of the SAC or the pharmaceutically or
sitologically acceptable salt thereof may be used as an active
ingredient for the therapeutic composition according to the present
embodiment.
[0053] The SAC used herein may be prepared from a plant belonging
to the Allium genus such as garlic, elephant garlic, onion,
orscallion using a method disclosed in European Patent Publication
No. EP 0429080A1, synthesis, fermentation, or any other known
method.
[0054] The SAC, the pharmaceutically or sitologically acceptable
salt of SAC, or a solvate or hydrate thereof, as an active
ingredient, may be directly administered to patients. However, a
composition including one or more of the active ingredients may be
administered or a combined formulation prepared by mixing the
active ingredients with an anti-Helicobacter pylori agent or a drug
for treating gastrointestinal disorders may also be administered to
patients.
[0055] The present invention provides a pharmaceutical composition
formulated into a formulation for oral administration, a
formulation for mucosal administration, an injection formulation, a
formulation for inhalation, and a formulation for external
application, but the formulation is not limited thereto. The
formulation for oral administration may include hard and soft
capsules, tablets, suspensions, powders, suspended-release
formulations, enteric formulations, granules, oleosacchara, fine
granules, pills, extracts, liquids, aromatic waters, emulsions,
syrups, elixirs, fluid extracts, infusodecoctions, tinctures,
medicated spirits, and infused oils, but is not limited thereto.
The formulation for mucosal administration may be troches, buccal
tablets, sublingual tablets, suppositories, and intranasal sprays,
but is not limited thereto. The injection formulation may be
subcutaneous injections, intramuscular injections, intravenous
injections, and implant tablets, but is not limited thereto. The
formulation for external application may be nasal drops, ophthalmic
solutions, otic solutions, ophthalmic ointments, pastes,
cataplasma, liniments, lotions, sprays, dusting powder, and liquids
for external use, but is not limited thereto.
[0056] The formulation according to the present embodiment may
further include one or more inert carriers, in addition to one or
more active ingredients, for example, excipients such as starch,
lactose, carboxymethyl cellulose, and kaolin, binders such as
water, gelatin, alcohol, glucose, gum Arabic, and gum Tragacanth,
disintegrants such as starch, dextrin, and sodium alginate,
lubricants such as talc, stearic acid, magnesium stearate, and
liquid paraffin, and other additives such as solubilizing
agents.
[0057] A daily dose of SAC may vary according to various factors
such as severity of disease, onset of the disease and a patient's
age, condition, and complications. In general, a daily dose of SAC
for an adult may be in the range of 1 mg to 10 g, preferably 100 mg
to 4 g, and more preferably 200 to 2,000 mg. However, the daily
dose may further increase for patients having severe symptoms or
complications to improve therapeutic efficiency. The formulations
may be administered in a single dose or divided into doses
administered 2 or 3 times per day. For example, one or two unit
dose formulations, each containing 200 to 500 mg of SAC may be
orally administered once or twice per day, but the administration
may be adjusted if required.
[0058] If the composition according to the present embodiment is a
food composition, the amount of the active ingredients may be
adjusted, if desired, for example, as disease-preventing or
treating food or health supplements. In general, the amount of SAC
in food or beverage may be in the range of 0.0001 to 90% by weight,
preferably 0.1 to 50% by weight of the total food or beverage. Even
though the amount of SAC in health supplements may be within the
range described above for long-term use, it may be increased since
the active ingredient is safe. Food including the food composition
according to the present embodiment may be meat, sausage, bread,
chocolate, candy, snack, pizza, instant noodle, gum, dairy
products, soup, beverages, tea, drinks, alcohol, and vitamins, but
is not limited thereto. Hereinafter, one or more embodiments will
be described in detail with reference to the following examples.
However, these examples are not intended to limit the purpose and
scope of the invention.
EXPERIMENTAL EXAMPLES
I. Effect of SAC in Animal Infected with Helicobacter pylori
[0059] Test Material
[0060] S-allyl-L-Cysteine (SAC) was purchased from TCI Chemical Co.
(Tokyo, Japan). Helicobacter pylori was American Type Culture
Collection (ATCC) 43504 (cagA+, vacA s1-ml type) and cultured in a
Mueller Hinton-Agar broth at 37.degree. C. for 48 hours, under 5%
CO.sub.2 microaerophilic conditions with a concentration of
1.times.10.sup.9 CFU/ml.
[0061] Test Animal
[0062] 8-week old male specific pathogen free (SPF) C57BL/6 mice
were used. Weights of the mice were measured in the Animal Lab,
Department of Pathology, the College of Veterinary Medicine,
Kyungbook National University. Then, the mice were classified into
4 groups and bred such that average weights of each group are
similar to each other. The mice were acclimated and bred in the
Animal Lab, Department of Pathology, the College of Veterinary
Medicine, Kyungbook National University at a temperature of
22.+-.3.degree. C., at a relative humidity of 50.+-.10%, with light
for 12 hours (light turned on at 08:00 and light turned off at
20:00) by an automatic temperature and humidity control system.
Other environments for breeding which might influence the test for
the entire test period were not considered for use. The mice were
given free access to a solid laboratory diet (PMI Nutrition
International, 505 North 4th Street Richmond, Ind. 47374, USA) and
filtered tap water with water bottles.
[0063] Test Group and Administration
[0064] 8-week old male C57BL/6 mice were classified into 4 groups,
i.e., a positive control group (to which Helicobacter pylori was
administered; PC), a negative control group (to which saline was
administered; NC), experimental group 1 (to which Helicobacter
pylori and 200 mg/kg of SAC were administered; SAC1), and
experimental group 2 (to which Helicobacter pylori and 400 mg/kg of
SAC were administered; SAC2). Each group had 10 mice. White
powdered SAC was diluted in tap water to concentrations of 20 mg/mL
and 40 mg/mL and 10 .mu.l/g (body weight) of the diluted SAC was
orally administered to the mice of the SAC1 and SAC2 groups three
times a week for 10 weeks. The mice were given free access to tap
water for the entire test period. The Helicobacter pylori was
collected with saline to a concentration of 1.times.10.sup.9
CFU/mL, and 0.2 mL of the diluted Helicobacter pylori was orally
administered to each mouse for 8 weeks from 2 weeks after SAC
administration. The mice were fasted for 8 hours before the
infection, and 0.15 mL of 0.2 M sodium bicarbonate (NaHCO.sub.3)
was administered to each mouse 10 minutes before the administration
of Helicobacter pylori in order to neutralize acidified stomach due
to the fasting. The same amount of saline instead of the infectious
substances was administered into the NC group. The mice of all
groups were fed with a normal diet. After a 10-week test period,
autopsies were performed on all mice, and samples of blood and
internal organs were collected for tissue pathological analyses.
The test was performed according to the following process.
[0065] Statistical Method
[0066] Statistical significance of the obtained data was tested
using an independent sample t-test. Statistical analysis was
performed using SPSS 14.OK, and a p-value of less than 0.05 was
considered significant.
Experimental Example 1
Effect of SAC on Weight
[0067] Weights of male C57BL/6 mice infected with Helicobacter
pylori were measured three times a week for the entire 10-week test
period to observe weight changes of the mice. The weights of the
mice gradually increased in all groups, except that the weights
were slightly and temporarily reduced at the time of Helicobacter
pylori infection and acquisition of serum to identify infection
(FIG. 1). A weight increase rate of the NC group was 31.3%, and
those of the PC group, SAC1 group, and SAC2 group were respectively
28.9%, 26.9%, and 28.7%. The weight increase rate of the PC group
was less than that of the NC group by 2.4%, the weight increase
rate of the SAC1 group was less than the PC group by 2%, and the
weight increase rate of the SAC2 group was less than that of the PC
group by 0.2%. Even though the weight increase rate of the SAC2
group was greater than that of the SAC1 group by 1.8%, this result
is deemed to be insignificant, and thus the Helicobacter pylori
infection and test materials did not significantly influence the
weight changes.
Experimental Example 2
Effect of SAC on Serum Anti-Helicobacter pylori (anti-H. pylori
IgG) Antibody-Formationcapability
[0068] Method
[0069] Enzyme-linked immunosorbent assay (ELISA) was used in order
to identify the effect of SAC on serum anti-Helicobacter pylori
antibody-formation capability. Orally infectious H. pylori ATCC
43504 and a recombinant toxin VacA specifically producing
Helicobacter pylori were used as antigens in mice. They were added
to a 96-well microplate for analysis to concentrations of 1
.mu.g/well and 10 ng/well, respectively, and the micro plate was
maintained at 4.degree. C. for coating. A supernatant was removed
and a blocking buffer (1% skim milk) was added to the micro plate
to inhibit unnecessary reactions, and the micro plate was
maintained at 37.degree. C. for 1 hour. 10 .mu.l of serum of mice
of all groups were added thereto and maintained at 37.degree. C.
for 2 hours. The microplate was washed with a Tris buffer solution
including Tween20, and anti-mouse IgG, as a secondary antibody,
bound to horse radish peroxidase (HRP) was added to the micro
plate. Then, the microplate was maintained at 37.degree. C. for 1
hour. Then, after being washed in the same manner, 100 .mu.l of a
mixture including a chromophore reagent of
3,3',5,5'-tetramethylbenzidine (TMB) and the same amount of
H.sub.2O.sub.2 was added thereto and the micro plate was maintained
in dark conditions. As a result, coloring was identified within 30
minutes. 100 .mu.l of 0.2 M sulfuric acid was added thereto to
terminate the reaction and absorbance was measured at 450 nm.
[0070] Result
[0071] As a result of measuring an anti-H. pylori IgG antibody
levelin serum, it was identified that the antibody against
Helicobacter pylori (anti-H. pylori IgG) was produced in the PC
group, the SAC1 group, and the SAC2 group to which Helicobacter
pylori were administered and was not produced in the NC group to
which Helicobacter pylori was not administered (FIGS. 2A and 2B).
Meanwhile, an antibody titer of anti-H. pylori IgG in the SAC1 and
SAC2 groups was less than that in the PC group, and the antibody
titer in the SAC2 group was significantly less than that in the
SAC1 group. Accordingly, the production of the antibody against
Helicobacter pylori was inhibited in a SAC concentration-dependent
manner (FIG. 2A). In addition, the production of the antibody
against Helicobacter pylori-producing toxin VacA (anti-s1m1 VacA
IgG) was inhibited in the same manner as in the anti-H. pylori IgG
(FIG. 2B). As a result, it was identified that Helicobacter pylori
infection was inhibited by SAC in a concentration-dependent
manner.
Experimental Example 3
Effect of SAC on Serum TNF-.alpha.
[0072] Method
[0073] In order to observe the effect of SAC on an inflammation
factor of TNF-.alpha. in serum, a commercially available mouse
TNF-.alpha. antibody analysis kit Microplate (R&D systems Inc.,
USA) was used and concentrations of TNF-.alpha. in serum of mice
were measured according to the manuals of the kit.
[0074] Result
[0075] The TNF-.alpha. value was increased in Helicobacter
pylori-administered groups when compared to the NC group
(p<0.1). The TNF-.alpha. of the SAC1 and SAC2 groups was less
than that of the PC group (p<0.09) (FIG. 3). As a result, it was
identified that SAC inhibits inflammation caused by Helicobacter
pylori infection in mice.
Experimental Example 4
Effect of SAC on Histological Change of Stomach Caused by
Helicobacter pylori
[0076] Method
[0077] Among lesions caused by Helicobacter pylori infection and
occurring for the 8 week-test period, tissue pathological changes
of a stomach were observed in a male C57BL/6 mouse Helicobacter
pylori infection model. A stomach sample was fixed with 10%
formalin and a paraffin block was formed. The paraffin block was
cut into sections having a thickness of 4 .mu.m, and the section
were stained with hematoxylin-eosin (H&E staining) and observed
using an optical microscope. All pathological features such as the
severity of cell lesions, the number of infiltrated eosinophils,
and the number of mitotic figures in the entire stomach were
double-checked. In addition, the number of eosinophils was checked
from a region where the esophagus and stomach join and counted from
three regions of the stomach, i.e., cardia, gastric pit, and
gastric crypt of lamina propria. The number of mitotic figures was
counted from two regions selected from the entire antrum from
cardia to pylorus. Thus two samples were obtained from each animal
and evaluated with a 400.times. magnification, and an average value
of each group was calculated.
[0078] Result
[0079] As a result of H&E staining, the infiltration of
eosinophils and the number of the mitotic figures were observed in
all groups. A number of eosinophils were found in lamina propria of
the stomach and eosinophils infiltrated into gastric mucosal
epithelium was observed (FIG. 4). The number of eosinophils was
significantly increased in the Helicobacter pylori-infected groups
compared to the NC group (p<0.01, FIG. 5B). On the other hand,
the number of eosinophils in the SAC1 and SAC2 groups was less than
that of the PC group, and particularly, the number of eosinophilsin
SAC1 group was significantly less than that of the PC group
(p<0.05, FIG. 5B). The number of the mitotic figures was also
increased in the Helicobacter pylori-infected groups compared to
the NC group. Even though there was no significant difference among
the PC group, the SAC1 group, and the SAC2 group, the number of the
mitotic figures was slightly reduced in the SAC2 group. The
increase of eosinophil infiltration in the stomach, caused by
Helicobacter pylori infection, has been reported in diverse
previous research. The reduction in eosinophil infiltration by SAC
indicates that SAC has protective effects against gastric lesions
caused by Helicobacter pylori infection.
Experimental Example 5
Effect of SAC on Serologic Parameters in Helicobacter
pylori-Infected Mouse Model
[0080] As a result of analyzing serum glutamic oxaloacetic
transaminase (GOT) that is an index related to general lesions, the
PC group exhibited the highest GOT level. Even though the SAC1 and
SAC2 groups were not significantly different from the PC group, the
GOT levels of the SAC1 and SAC2 groups were less than that of the
PC group (p<0.08, FIG. 7).
[0081] As a result of analyzing serum glutamate pyruvate
transaminase (GPT) that is an index related to liver lesion, the PC
group exhibited the highest GPT level. Even though the SAC1 and
SAC2 groups were not significantly difference from the PC group,
the GOT level of the SAC1 and SAC2 groups was less than that of the
PC group (FIG. 8).
Experimental Example 6
Measurement of Serum Cu/Zn-SOD Level
[0082] Method
[0083] Effect of SAC on copper and zinc containing-superoxide
dismutase (Cu/Zn-SOD) that is an antioxidant enzyme in serum was
observed in a male C57BL/6 mouse Helicobacter pylori infection
model for the entire 10-week test period. In this regard, a
superoxide dismutase activity assay kit (BioVision, Mountain View,
Calif., USA) was used. 20 .mu.l of serum and 200 .mu.l of a WST
working solution as a substrate were mixed. 20 .mu.l of an enzyme
working solution was added thereto, and the mixture was maintained
at 37.degree. C. for 20 minutes. Absorbance was measured at 450 nm.
A control reaction was conducted in the same manner as described
above, except that 20 .mu.l of distilled water was added thereto
instead of serum. SOD activity (inhibition rate %) was
calculated.
[0084] Result
[0085] The Cu/Zn-SOD levels of the SAC1 and SAC2 groups were
greater than that of the PC group by about 4% and about 3%,
respectively. It was observed that the Cu/Zn-SOD levels of the SAC2
group were slightly less than the SAC1 group. Accordingly, it was
identified that SAC promotes expression of SOD that is produced by
a defense mechanism against Helicobacter pylori infection.
II. Gastric Mucosal Protective Effect of SAC in Animals Having
Gastric Lesions Caused by Drugs
[0086] The gastric mucosal protective effect of SAC was evaluated
in animals having gastric lesions induced by hydrochloric
acid-ethanol, aspirin, or indomethacin.
[0087] Test Material
[0088] SAC was purchased from TCI Chemical Co. (Tokyo, Japan).
Sterile water for injection (Model No. 73H5F21, Dae Han
Pharmaceutical Co. Ltd.) was used as a vehicle, and Stillen.RTM.
was used as a positive control material. 0.5% CMC-Na and sterile
water for injection were used as excipients for Stillen.RTM..
Hydrochloric acid was purchased from Samjung Chemical, Co., ethanol
was purchased from Baker, Co., aspirin was purchased from Sigma,
Co., and indomethacin was purchased from Sigma, Co.
[0089] Test Animals
[0090] 7 to 8-week old male specific pathogen free (SPF)
HsdKoat:Sprague-Dawley.RTM..TM. SD.RTM..TM. rats (weight of 7-week
old males was in the range of 208.44 to 227.39 g, and weight of
8-week old males was in the range of 223.85 to 245.03 g, purchased
from Koatech, Co. Ltd., Gyunggido, Korea) were quarantined and
acclimated in the Animal Lab for 7 days. The rats were bred at a
temperature of 23 3.degree. C., at a relative humidity of 55 15%,
with light for 12 hours (light turned on at 08:00 and light turned
off at 20:00) while air was ventilated 10 to 20 times/hr in the
Animal Experiment Lab of Gyeonggi Bio-Center. Other environments
for breeding which might influence the test for the entire test
period were not considered for use. The rats were given free access
to a solid laboratory diet (Harlan Co. Ltd., USA. Teklad certified
global 18% protein rodent diet, 2918C) that was supplied by Folas
International. According to the analysis of certificate of diet
composition, there were no ingredients or contaminants that could
have had an adverse effect on the test. The rats were given free
access to tap water that was sterilized using a UV sterilizer and a
micro filter with water bottles.
[0091] Test Group and Administration
[0092] The rats were classified into a vehicle control group G1 to
which only a vehicle was administered, a experimental group G2 to
which 100 mg/kg of SAC was administered, a experimental group G3 to
which 200 mg/kg of SAC was administered, a experimental group G4 to
which 400 mg/kg of SAC was administered, and a positive control
group G5 to which 100 mg/kg of Stillen.RTM. (55.6 mg/kg as an
active ingredient) as a positive control material were
administered. Each group included 8 rats in a hydrochloric
acid-ethanol-induced animal model (Experiment Example 7) and
included 6 rats in an aspirin-induced animal model (Experimental
Example 8) and in an indomethacin-induced animal model
(Experimental Example 9).
TABLE-US-00001 TABLE 1 Total Adminis- Number of Number of tered
Vol- Dose Group Gender animals animals ume(ml/kg) (mg/kg) G1 Male
8(6) 1~8(1~6) 10 0 G2 Male 8(6) 9~16(7~12) 10 100 G3 Male 8(6)
17~24(13~18) 10 200 G4 Male 8(6) 25~32(19~24) 10 400 G5 Male 8(6)
33~40(25~30) 10 55.6.sup.a) G1: Vehicle control group G2 to G4:
Experimental groups to which SAC is administered G5: Positive
control group to which a positive control material is administered
.sup.a)Dose of active ingredient
[0093] The test substance was directly administered to the stomach
using an injection tube equipped with a sonde for oral
administration in a single dose once per day.
[0094] No animals died and no other changes were observed in any of
the groups, and significant weight change with respect to the
administration of the SAC was not observed at the administration
and for the entire test period.
[0095] Statistical Method
[0096] Comparisons of the vehicle control group with the
experimental groups and the positive control material-administered
experimental group were verified using one-way analysis of variance
(One-way ANOVA). In this regard, significance and homoscedasticity
were accepted, and thus a post-doc test was conducted using a
Duncantest. Significance was accepted when p<0.05, and SPSS 10.1
was used.
Experimental Example 7
Effect of SAC on Lengths of Gastric Lesions and Gastric Lesion
Inhibiting Rate in Hydrochloric Acid-ethanol-induced Gastric Lesion
Animal Model
[0097] Method
[0098] The test substances were administered. After one hour, 1.5
ml of 150 mM HCl in 60% ethanol was orally administered to each
rat. The rats to which ethanol and SAC were administered were
fasted without water in stainless steel breeding cages for 1 hour.
After one hour from the hydrochloric acid-ethanol administration,
the rats were sacrificed under anesthesia using ether and their
stomachs including parts of the duodenum and esophagus were
isolated. The insides of the stomachs were immediately washed with
13 ml of a 2% neutral buffered formalin, and the duodenum and
esophagus parts were fixed with forceps. Then, 13 ml of the 2%
neutral buffered formalin was added thereto and maintained for 5
minutes for fixation. The greater curvature of each stomach was
incised, fixed to a dissection table, and unfolded to measure the
lengths of gastric lesions using vernier calipers. Photographs of
the unfolded stomachs were taken (FIG. 12) and the stomachs were
fixed with a 10% neutral buffered formalin.
[0099] Result
[0100] According to this gastric lesion model, ethanol directly
stimulates gastric mucosa, induces edema in a muscle layer under a
mucous membrane to cause a transient ischemic condition, and thus
cell necrosis is induced due to oxidative damage, and hydrochloric
acid directly stimulates the gastric mucosa and accelerates gastric
motility to cause acute gastritis. By gross finding, the lesion was
observed over the entire gastric mucosa and hemorrhage was observed
in a long line. After one hour from the hydrochloric acid-ethanol
administration, the rats of the vehicle control group had lesions
over the entire gastric mucosa which was identified by atopsy
(209.60.+-.28.39 mm). The experimental group to which 200 mg/kg of
SAC was administered (106.65.+-.16.70 mm, p<0.01), the
experimental group to which 400 mg/kg of SAC was administered
(72.25.+-.19.33 mm, p<0.01), and the positive control group to
which Stillen.RTM. was administered (102.51.+-.11.35, p<0.01)
exhibited significantly less lesions than the vehicle control group
(refers to FIGS. 10 and 12).
Gastric lesion inhibiting rate (%)=(average length of the vehicle
control group-length of gastric lesion of each animal)/average
length of the vehicle control group.times.100.
The experimental group to which 200 mg/kg of SAC was administered
(41.12.+-.7.97%, p<0.01), the experimental group to which 400
mg/kg of SAC was administered (65.53.+-.9.22%, p<0.01), and the
positive control group to which Stillen.RTM. was administered
(51.09.+-.5.41%, p<0.01) exhibited a significantly greater
gastric lesion inhibiting rate (%) than the vehicle control group
(refers to FIG. 11).
Experimental Example 8
Effect of SAC on Area of Gastric Lesion and Gastric Lesion
Inhibiting Rate in Aspirin-induced Gastric Lesion Animal Model
[0101] Method
[0102] The rats were fasted for more than 24 hours in a normal
environment, the test substance was administered, and 200 mg/kg of
aspirin in 0.15 mol/L HCl was orally administered after 30 minutes.
After three hours from the aspirin administration, the rats were
sacrificed under ether anesthesia and their stomachs including
parts of the duodenum and esophagus were isolated. The stomachs
were was fixed for 10 minutes by injecting 12 ml of 2% formalin.
The greater curvature of each stomach was incised and unfolded,
photographs of the gastric grandular region were taken, and then
the areas of the lesions were measured using an image analyzer.
[0103] Result
TABLE-US-00002 TABLE 2 Animal number (n = 6) UlcerArea Group 1 2 3
4 5 6 (mm.sup.2) SD IR (%) G1 165.76 1555.77 216.14 231.88 285.72
236.67 215.3 48.35 G2 91.68 44.12 40.95 63.52 63.52 64.67 68.6
25.94 -68.2 G3 46.29 19.37 5.41 27.07 27.07 43.86 31.4 16.99 -85.4
G4 63.26 9.34 70.28 40.03 40.03 11.58 32.5 29.78 -84.9 G5 16.86
29.58 7.82 71.91 71.91 61.96 32.5 28.09 -84.9
[0104] According to this gastric lesion model, aspirin that is a
nonsteroidal anti-inflammatory drug inhibits the synthesis of
prostaglandin that protects stomach walls, thereby causing gastric
ulcers. The lesions were observed over the entire gastric mucosa
and bleeding was observed in a net form. After aspirin
administration, the rats of the vehicle control group had bleedings
and lesions over the entire gastric mucosa which was identified by
autopsy (215.3.+-.48.35 mm.sup.2). The experimental group to which
100 mg/kg of SAC was administered (68.6.+-.25.94 mm.sup.2), the
experimental group to which 200 mg/kg of SAC was administered
(31.4.+-.16.99 mm.sup.2), the experimental group to which 400 mg/kg
of SAC was administered (32.5.+-.29.78 mm.sup.2), and the positive
control group (32.5.+-.28.09 mm.sup.2) exhibited significantly less
lesions than the vehicle control group (refers to FIGS. 13 and
15).
Gastric lesion inhibiting rate (%)=(average area of the vehicle
control group-area of gastric lesion of each animal)/average area
of the vehicle control group.times.100.
The experimental group to which 100 mg/kg of SAC was administered
(68.2%), the experimental group to which 200 mg/kg of SAC was
administered (85.4%), the experimental group to which 400 mg/kg of
SAC was administered (84.9%), and the positive control group
(84.9%) exhibited a significantly greater gastric lesion inhibiting
rate (%) than the vehicle control group (refer to FIG. 14).
Experimental Example 9
Effect of SAC on Area of Gastric Lesion and Gastric Lesion
Inhibiting Rate in Indomethacin-induced Gastric Lesion Animal
Model
[0105] Method
[0106] The rats were fasted for more than 24 hours in a normal
environment, SAC was administered, and 25 mg/kg of indomethacin in
distilled water was orally administered after 30 minutes. After six
hours from the indomethacin administration, the rats were
sacrificed under ether anesthesia and their stomachs including
parts of the duodenum and esophagus were isolated. The stomachs
were fixed for 10 minutes by injecting 12 ml of 2% formalin, the
greater curvature of each stomach was incised and unfolded,
photographs of the lesions were taken, and then the areas of the
lesions were measured using an image analyzer.
[0107] Result
TABLE-US-00003 TABLE 3 Animal number (n = 6) UlcerArea Group 1 2 3
4 5 6 (mm.sup.2) SD IR (%) G1 6.4 18.0 6.7 4.2 0.9 9.6 7.6 5.85 G2
1.3 1.6 1.5 9.4 -- 2.3 3.2 3.47 -57.9 G3 0.0 0.3 0.9 0.6 0.7 0.4
0.5 0.30 -93.8 G4 0.8 0.4 0.1 0.2 0.1 1.0 0.4 0.36 -94.4 G5 3.7 0.6
2.7 0.7 7.7 5.2 3.4 2.75 -55.2
[0108] According to this gastric lesion model, indomethacin that is
a nonsteroidal anti-inflammatory drug inhibits the synthesis of
prostaglandin that protects stomach walls, thereby causing gastric
lesions. Local lesions were observed in the gastric mucosa and
bleeding was observed. After indomethacin administration, the rats
of the vehicle control group had lesions having an area of
7.6.+-.5.85 mm.sup.2 which was identified by autopsy. In the
experimental group to which 100 mg/kg of SAC was administered, the
area of the lesions was 3.2.+-.3.47mm.sup.2 which was less than
that of the vehicle control group by a half or more. The
experimental group to which 200 mg/kg of SAC was administered
(0.5.+-.0.30 mm.sup.2), the experimental group to which 400 mg/kg
of SAC was administered (0.4.+-.0.36 mm.sup.2), and the positive
control group (3.4.+-.2.75 mm.sup.2) exhibited significantly less
lesions than the vehicle control group. In particular, the
experimental groups to which 200 mg/kg and 400 mg/kg of SAC were
administered exhibited light gastric lesions and no bleedings
(FIGS. 16 and 18).
[0109] The gastric lesion inhibiting rate (%) was calculated in the
same manner as in Experimental Example 8. The experimental group to
which 100 mg/kg of SAC was administered (57.9%), the experimental
group to which 200 mg/kg of SAC was administered (93.8%), the
experimental group to which 400 mg/kg of SAC was administered
(94.4%), and the positive control group (55.2%) exhibited a
significantly greater gastric lesion inhibiting rate (%) than the
vehicle control group. Particularly, it was identified that the
gastric lesion may be completely inhibited if 200 mg/kg or more of
SAC is administered (refers to FIG. 17).
PREPARATION EXAMPLES
[0110] Various formulations including SAC as an active ingredient
were prepared as follows.
Preparation Example 1
Manufacture of Tablet
[0111] SAC 200 mg
[0112] Lactose 50 mg
[0113] Starch 10 mg
[0114] Magnesium stearate appropriate quantity
[0115] The ingredients above were mixed and tableted using a known
method to prepare a tablet.
Preparation Example 2
Manufacture of Powder
[0116] SAC 250 mg
[0117] Lactose 30 mg
[0118] Starch 20 mg
[0119] Magnesium stearate appropriate quantity
[0120] The ingredients above were mixed and filled in a chartula
coated with polyethylene, and the chartula was sealed to prepare
powder.
Preparation Example 3
Manufacture of Capsule
[0121] SAC 500 mg
[0122] Lactose 30 mg
[0123] Starch 28 mg
[0124] Magnesium stearate appropriate quantity
[0125] The ingredients above were mixed and filled in a gelatin
hard capsule using a known method to prepare a capsule.
Preparation Example 4
Manufacture of Suspension
[0126] SAC 50 mg
[0127] Isomerized sugar 10 g
[0128] Sugar 30 mg
[0129] Sodium carboxymethyl cellulose 100 mg
[0130] Lemon flavor appropriate quantity
[0131] Total volume including purified water 100 ml
[0132] The ingredients above were mixed to prepare a suspension
using a known method and the suspension was filled in a 100
ml-brown bottle and sterilized.
Preparation Example 5
Manufacture of Soft Capsule (Amount in One Soft Capsule)
[0133] SAC 500 mg
[0134] Polyethylene glycol 400 400 mg
[0135] Concentrated glycerin 55 mg
[0136] Purified water 35 mg
[0137] Polyethylene glycol and concentrated glycerin were mixed,
and purified water was added thereto. Flavone was added thereto
while the mixture was maintained at about 60.degree. C., and the
mixture was stirred at about 1,500 rpm with a stirrer. The mixture
was cooled to room temperature while slowly stirring and bubbles
were removed using a vacuum pump to prepare contents for a soft
capsule. The coating of the soft capsule was prepared using gelatin
and a plasticizer which are known in the art. 132 mg of gelatin, 52
mg of concentrated glycerin, 6 mg of 70% disobitol solution, an
appropriate amount of ethyl vanillin as a flavoring agent, and
carnauba wax as a coating base were used to prepare one soft
capsule using a known method.
Preparation Example 6
Manufacture of Injection
[0138] SAC 200 mg
[0139] Mannitol 180 mg
[0140] Sterilized distilled water for injection 2974 mg
[0141] Na.sub.2HPO.sub.412H.sub.2O 26 mg
[0142] An ample having the ingredients above was prepared using a
known method.
Preparation Example 7
Manufacture of Beverage
[0143] SAC 0.01 g
[0144] Citric acid 8.5 g
[0145] White sugar 10 g
[0146] Glucose 2.5 g
[0147] DL-malic acid 0.3 g
[0148] Purified water appropriate quantity
[0149] The ingredients above and an appropriate amount of purified
water were mixed to a total volume of 100 mL and stirred to prepare
a beverage using a known method.
[0150] While the present invention has been particularly shown and
described with reference to exemplary embodiments thereof, it will
be understood by those of ordinary skill in the art that various
changes in form and details may be made therein without departing
from the spirit and scope of the present invention as defined by
the following claims.
* * * * *